Evolution directed

Directed evolution is an iterative process that mimics the natural evolution process in vitro, by generating a diverse library of enzymes and selecting those with the desired features. Natural evolution is very effective in the long term (bacteria adapt to every environment, living even in so-called black smokers, deep-ocean vents where temperatures can reach 350°C and the pressure is 200bar [93]). Unfortunately, it typically takes millions of years. Happily, directed evolution can be carried out within weeks or months and with an unlimited number of parents. Importantly, and unlike rational design, directed evolution is a stochastic method. It does not require any structural or mechanistic information on the enzyme of interest (although such information can help). 

Evolving enzymes with specific properties (e.g., higher activity in organic solvents) is basically a search process in the enzyme space (see Chapter 6 for a more detailed discussion). Onereasonwhy directed evolution outperforms rational design is the sheer size of this space. Since enzymes are made of 20 amino adds, even the sequence of a short enzyme containing just 80 amino acids can already have 2080 permutations. 

This is far more than the number of atoms that make up the Earth. It is also the reason why directing the evolution of an existing enzyme is much likelier to succeed than searching for catalytically active amino add sequences in random peptide libraries. 

With the advances in genetic engineering technology, directed evolution is no longer rocket science. It is a proven technology which opens a fast and relatively inexpensive pathway for developing new biocatalysts [97]. Successful synthetic 

In this section, we explore the theoretical basis for each process in directed evolution mutation, recombination, and screening. Although this section is subdivided to discuss each process separately, the evolutionary parameters cannot be treated entirely independently. For example, 

Because the landscape of real proteins is unknown, most of the results we describe in Section III rely on assumptions discussed in Section II. The results are presented for a range of different theoretical landscapes—for example, the random energy model and the uncoupled case—with the assumptions that the real protein landscape lies between these bounds and can be described statistically. Determining the most effective combination of parameters, adjusting them according to the landscape fea- 

The fitness landscape also affects the reproducibility of the experiment. If the landscape is very smooth, then there are many possible paths between two sequences (Wang et al., 1996). However, as the ruggedness of the landscape increases, the number of paths sharply decreases. 

Therefore, the walk length before a local optimum is reached will be longer for an adaptive walk than a steepest ascent walk. The walk length R for an adaptive and steepest ascent walk has been estimated using the NK-model  

In a similar study, protein evolution has been analyzed using the random energy model (Macken and Perelson, 1989). Macken and Perel-son calculate the probability of a random walk taking k steps to a local optimum as 

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Combinatorial methods are often referred to as in vitro or directed evolution techniques. In nature, the random DNA mutations that lead to changes in protein sequences occur rarely and so evolution is usually a slow... 

Natural selection works through the complementary processes of mutation and genetic reassortment by recombination. The oligonucleotide-directed mutagenesis methods used in the foregoing examples do not allow for recombination instead, mutations are combined manually to optimize a protein sequence. Willem Stemmer at Maxygen invented a method of directed evolution that uses both mutation and recombination. This method, called... 

Crameri, A., Raillard, S.-A., Bermudez, E., Stemmer, W.P.C. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391 288-291, 1998. 

Directed Evolution as a Means to Engineer Enantioselective Enzymes... 

Figure 2.1 Strategy for directed evolution of an enantioselective enzyme [6,8].

Figure 2.2 The experimental stages of directed evolution of enantioselective enzymes [6,8].

A number of high-throughput enantiomeric excess assays have been developed, yet none are completely general. This crucial aspect of directed evolution of... 

Examples of Enhancing the Enantioselectivity of Enzymes by Directed Evolution... 

These initial systematic studies regarding the directed evolution of PAL allowed several conclusions to be made. Protein sequence space can be explored successfully by applying the following strategies [8c,33j ... 

Figure 2.9 Schematic summary of the directed evolution of enantioselective lipase variants originating from the WT PAL used as catalysts in the hydrolytic kinetic resolution of ester rac-1. CMCM = Combinatorial multiple-cassette mutagenesis [8c,22],...

The data in Figure 2.12 are the results of initial mutagenesis experiments, which does not yet constitute directed evolution. An evolutionary process was subsequently induced by combining the mutations of two improved mutants of the first round [46]. Thereby new mutants were obtained, which show an increase of activity relative to the WT by more than 2 orders of magnitude. Although enantioselectivity was not the... 

The Bacillus subtilis lipase A (BSLA) was the subject of two short directed evolution studies [19,47]. In one case systematic saturation mutagenesis at all of the ISlpositions of BSLA was performed [19]. Using meso-l,4-diacetoxy-2-cyclopentene as the substrate, reversed enantioselectivity of up to 83% ee was observed. In another study synthetic shuffling (Assembly of Designed Oligonucleotides) was tested using BSLA [47]. 

Esterases have a catalytic function and mechanism similar to those of lipases, but some structural aspects and the nature of substrates differ [4]. One can expect that the lessons learned from the directed evolution of lipases also apply to esterases. However, few efforts have been made in the directed evolution of enantioselective esterases, although previous work by Arnold had shown that the activity of esterases as catalysts in the hydrolysis of achiral esters can be enhanced [49]. An example regarding enantioselectivity involves the hydrolytic kinetic resolution of racemic esters catalyzed by Pseudomonasfluorescens esterase (PFE) [50]. Using a mutator strain and by screening very small libraries, low improvement in enantioselectivity was... 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, which catalyze the hydrolysis of hydantoins [4,54]. As synthetic hydantoins are readily accessible by a variety of chemical syntheses, including Strecker reactions, enantioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, hydantoins are easily racemized chemically or enzymatically by appropriate racemases, so that dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases using WT hydantoinases have been reported [54]. However, if asymmetric induction is poor or ifinversion ofenantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of i-methionine in a whole-cell system ( . coli) (Figure 2.13) [55]. 

Nitrilases catalyze the synthetically important hydrolysis of nitriles with formation of the corresponding carboxylic acids [4]. Scientists at Diversa expanded the collection of nitrilases by metagenome panning [56]. Nevertheless, in numerous cases the usual limitations of enzyme catalysis become visible, including poor or only moderate enantioselectivity, limited activity (substrate acceptance), and/or product inhibition. Diversa also reported the first example of the directed evolution of an enantioselective nitrilase [20]. An additional limitation had to be overcome, which is sometimes ignored, when enzymes are used as catalysts in synthetic organic chemistry product inhibition and/or decreased enantioselectivity at high substrate concentrations [20]. 

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